An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

Integrated Omic Approaches Reveal Molecular Mechanisms of Tolerance during Soybean and Meloidogyne incognita Interactions.

The root-knot nematode (RKN), Meloidogyne incognita, is a devastating soybean pathogen worldwide. The use of resistant cultivars is the most effective method to prevent economic losses caused by RKNs. To elucidate the mechanisms involved in resistance to RKN, we determined the proteome and transcriptome profiles from roots of susceptible (BRS133) and highly tolerant (PI 595099) Glycine max genotypes 4, 12, and 30 days after RKN infestation. After in silico analysis, we described major defense molecules and mechanisms considered constitutive responses to nematode infestation, such as mTOR, PI3K-Akt, relaxin, and thermogenesis. The integrated data allowed us to identify protein families and metabolic pathways exclusively regulated in tolerant soybean genotypes. Among them, we highlighted the phenylpropanoid pathway as an early, robust, and systemic defense process capable of controlling M. incognita reproduction. Associated with this metabolic pathway, 29 differentially expressed genes encoding 11 different enzymes were identified, mainly from the flavonoid and derivative pathways. Based on differential expression in transcriptomic and proteomic data, as well as in the expression profile by RT-qPCR, and previous studies, we selected and overexpressed the GmPR10 gene in transgenic tobacco to assess its protective effect against M. incognita. Transgenic plants of the T2 generation showed up to 58% reduction in the M. incognita reproduction factor. Finally, data suggest that GmPR10 overexpression can be effective against the plant parasitic nematode M. incognita, but its mechanism of action remains unclear. These findings will help develop new engineered soybean genotypes with higher performance in response to RKN infections.

Effects of hydroalcoholic extract of Celtis iguanaea on markers of cardiovascular diseases and glucose metabolism in cholesterol-fed rats

ABSTRACT Celtis iguanaea (Jacq.) Sarg., Cannabaceae, is popularly used in the treatment of diabetes mellitus. However, chemical and pharmacological investigations are lacking. In this study, we investigated the effects of the hydroalcoholic extract from C. iguanaea on markers of cardiovascular diseases and the glucose metabolism in cholesterol-fed rats. Therefore, hypercholesterolemic rats (1% cholesterol) were orally treated with C. iguanaea extract (C-150, CI-300, or CI-600 mg/kg) or simvastatin (4 mg/kg) (n = 6) once a day (30 days) with a hypercholesterolemic diet. A control group (C) was given saline. C. iguanaea extract showed significant decreases in serum levels of total cholesterol, LDL-cholesterol, HMG-CoA-reductase, interleukin-1 and 6, TNF-α and IFN-γ when compared to group C (p < 0.001). Hypoglycemic effects were observed along with a decrease of the activity of sucrase (CI-600), maltase (CI-150, CI-300), and an increase in muscle glycogen levels (CI-300). Antioxidant effects were observed in plasma by the decrease of TBARS and increase of nonprotein thiols levels (CI-600). The histopathological analysis showed a significant decrease in the liver fat area for C. iguanaea extract compared to group C (p < 0.001). Our results suggest that the biological effects of C. iguanaea extract could be related to the flavonoids that possibly exert antioxidant, enzymatic inhibitory, and insulin-mimetic effects.